Genes containing a DNA sequence coding for hydroxynitrile lyase, recombinant proteins derived therefrom and having hydroxynitrile lyase activity, and use thereof

ABSTRACT

New genes containing a DNA sequence coding for hydroxynitrile lyase, which genes can be prepared via a primer combination based on the DNA sequence of of the 5′-region of the mdl genes from  Prunus serotina  and from  Prunus amygdalus  and/or a primer 2 based on the 3′-region of the DNA sequences of one of the hydroxynitrile lyase isoenzymes from  Prunus serotina  or from  Prunus amygdalus , subsequent amplification with a DNA polymerase using a DNA from organisms, containing genes coding for hydroxynitrile lyase, as templates and cloning, and also recombinant proteins which can be prepared in suitable host cells by heterologous expression of the DNA sequence of said HNL genes, and proteins and fusion proteins derived therefrom and use of said proteins for preparing (R)- or (S)-cyanohydrins.

[0001] New genes containing a DNA sequence coding for hydroxynitrilelyase, recombinant proteins derived therefrom and having hydroxynitrilelyase activity, and use thereof

[0002] Biocatalytic processes have become very important for thechemical industry. In this connection, carrying out chemical reactionswith the aid of biologic catalysts is particularly interesting in thosefields of application in which it is possible to utilize the frequentlyfound enzyme property of preferably converting or forming in chemicalreactions with chiral or prochiral components one of the twoenantiomers.

[0003] Essential requirements for utilizing said advantageous propertiesof enzymes are the availability of said enzymes in industrially requiredamounts and a sufficiently high reactivity and also stability under thereal conditions of an industrial process.

[0004] Cyanohydrins are a particularly interesting class of chiralchemical compounds. Cyanohydrins are important, for example, in thesynthesis of α-hydroxy acids, α-hydroxyketones, β-aminoalcohols whichare used for producing biologically active substances, for examplepharmaceutical active substances, vitamins or pyrethroid compounds.

[0005] Said cyanohydrins are prepared by addition of hydrocyanic acid tothe carbonyl group of a ketone or aldehyde.

[0006] Industrial production of chiral compounds such as, for example,(S)-cyanohydrins was made possible by making use of the enzyme(S)-hydroxynitrile lyase from Hevea brasiliensis and is described, forexample, in WO 97/03204, EP 0 951561 and EP 0 927 766.

[0007] However, there is a large variety of interesting chemicalcompounds for which the R enantiomers are important for industrialapplications. Up until now, only processes which can be used only on thelaboratory scale have been described for preparing a number of products(e.g.: EP 0 276 375, EP 0 326 063, EP 0 547 655). In this connection,mainly enzyme preparations obtained from plants of the Rosaceae family,for example from the kernels of almonds (Prunus amygdalus), were used.

[0008] Recently, Prunus species have become more and more important sothat attempts were made to investigate said species in greater detail.

[0009] The specialist literature, for example Plant Physiology, April1999, Vol 119, pp. 1535-1546, discloses that Prunus species can containa plurality of R-HNL isoenzymes. These isoenzymes are expressed atdifferent levels in various tissues of the plant. It was possible toidentify in the plant Prunus serotina which is a close relative ofPrunus amygdalus 5 different isoenzymes up until now and to sequencetheir genes. Only one Prunus amygdalus isoenzyme has been described upuntil now in Planta (1998) 206: 388-393, and this isoenzyme is moststrongly expressed in the flower bud. A gene for said R-HNL isoenzymehas already been isolated and the cDNA has been sequenced.

[0010] However, no successful (functional) heterologous expression ofsuch a gene has been reported in the specialist literature or patentliterature.

[0011] Industrial applications on a large scale, too, have not beencarried out up until now, the main reason being that enzyme preparationsfrom almond kernels with hydroxynitrile lyase activity have not beenavailable up until now in sufficient quantities and at justifiablecosts.

[0012] It was therefore an object of the invention to create a basiswhich can provide an R-hydroxynitrile lyase in amounts required forindustrial applications.

[0013] This object was achieved by looking for a way of producing anenzyme corresponding to the R-HNL preparation of Prunus amygdalus bygenetic engineering strategies with the aid of an appropriaterecombinant microorganism strain. Such a recombinant enzyme with R-HNLactivity ought to be made technically available in this way in asufficient amount.

[0014] It can be derived from the DNA sequences described in theliterature above that particular regions in the genes of the variousisoenzymes are highly conserved. According to the invention, this isused as a basis for generating primers for PCR amplification of P.amygdalus R-HNL. With the aid of such primers it is then possible, usingDNA isolated from for example almond kernels (P. amygdalus) as template,to amplify by means of PCR DNA pieces which show in the analysis byagarose gel electrophoresis distinct specific bands. According to theinvention, bands were found which correspond to the size of mdl genes(Planta (1998) 206: 388-393). Subsequently, appropriate primers for allisoenzymes known in Prunus serotina are generated and corresponding PCRproducts are obtained. DNA from this region is isolated from appropriatepreparative agarose gels and cloned into standard vectors for cloning ofPCR-generated fragments in Escherichia coli.

[0015] Sequence analysis of a series of selected clones revealed thepresence of clones with homologies to the particular, already knownR-HNL genes of Prunus species, although the sequences of the clones orgenes obtained in this way differ in several sequence positions from thesequences already known or published, whereby important functionaldifferences are established.

[0016] As a result, a new variant of HNL genes was unexpectedly found,although primer combinations which made use of the already knownsequence of a cDNA obtained from Prunus amygdalus flower material wereused.

[0017] The new genes were sequenced and the genomic DNA sequence wasdetermined.

[0018] Accordingly, the present invention relates to new genescontaining a DNA sequence coding for hydroxynitrile lyase, which genescan be prepared via a primer combination of a primer 1 based on the DNAsequence of the 5′-region of the mdl genes from Prunus serotina and fromPrunus amygdalus and/or a primer 2 based on the 3′-region of the DNAsequences of one of the hydroxynitrile lyase isoenzymes from Prunusserotina or from Prunus amygdalus, subsequent amplification with a DNApolymerase using a DNA from organisms, containing genes coding forhydroxynitrile lyase, as templates and cloning.

[0019] Thus it is possible, for example, to prepare gene-specific PCRprimer based on sequence homology of the Prunus amygdalus MDL1 gene andof the Prunus serotina mdl.5 gene, and, as a result, a new gene, theHNL5 gene, is obtained after amplification and cloning.

[0020] The Prunus amygdalus HNL5 gene produced by PCR amplification, forexample, has the nucleotide sequence depicted in FIG. 1, which islikewise a subject of the invention. The invention also relates to HNL5genes having a nucleotide sequence which is at least 80%, preferably85%, identical to the sequence depicted in FIG. 1.

[0021] The new HNL5 gene differs from the published sequence of thePrunus amygdalus MDL 1 gene in 7 base pairs.

[0022] Furthermore it is possible, for example, to prepare gene-specificPCR primers based on sequence homology of the Prunus serotina mdl1 gene,and, as a result, a new gene, the HNL1 gene, is obtained afteramplification and cloning.

[0023] The HNL1 gene produced by PCR amplification, for example, has thenucleotide sequence depicted in FIG. 8, which is likewise a subject ofthe invention. The invention also relates to HNL1 genes having anucleotide sequence which is at least 80%, preferably 85%, identical tothe sequence depicted in FIG. 8.

[0024] Analogously, it is possible, according to the invention, toprepare further gene-specific PCR primers, for example based on thesequence of the Prunus amygdalus MDL1 gene and/or based on the sequenceof the known Prunus serotina mdl1, mdl2, mdl3 and mdl4 genes, and thisresults in obtaining, after amplification and cloning, further new genessuch as, for example, HNL2, 3 or 4 which are all subjects of the presentinvention.

[0025] The genomic clones and genomic DNA thereof form the basis forobtaining enzyme preparations by heterologous expression, for example byinducible or constitutive expression, in various host cells.

[0026] Furthermore, sequence analysis of the genomic DNA of the newgenes of the invention shows that the proteins encoded by the new genespossess a signal sequence or a signal peptide, and this also makessecretory expression of heterologous proteins in suitable host cellspossible.

[0027] In this connection, specific expression vectors are used forexpressing the protein of one of the cloned new genes as a fusionprotein with a signal peptide.

[0028] Accordingly, the present invention further relates to recombinantproteins which can be prepared in suitable host cells by heterologousexpression of the genomic DNA sequence of the Prunus amygdalus HNL genes(for example HNL1, HNL2, HNL3, HNL4 and HNL5). Examples of suitable hostcells in this connection are microorganisms. Preference is given toeukaryotic microorganisms and particular preference is given to fungi.Examples are Saccharomyces cerevisiae or Pichia pastoris.

[0029] For example, the amino acid sequence of Prunus amygdalushydroxynitrile lyase HNL5, derived from the nucleotide sequence of theHNL5 gene, is depicted in FIG. 3 and the amino acid sequence of Prunusamygdalus hydroxynitrile lyase HNL1, derived from the nucleotidesequence of the HNL1 gene, is depicted in FIG. 9.

[0030] In addition, the invention relates to the use of a DNA sequencewhich codes for the signal peptide of a hydroxynitrile lyase, forexample of Rosacea species, for secretory expression of heterologousproteins in host cells and to the proteins obtained in this way.

[0031] Accordingly, the invention further relates to fusion proteins orheterologous proteins which can be prepared by using a DNA sequencewhich codes for the signal peptide of a hydroxynitrile lyase, forexample of Rosacea species, and by secretory expression of said DNAsequence in suitable host cells.

[0032] Examples of suitable host cells in this connection are againmicroorganisms. Preference is given to bacteria or eukaryoticmicroorganisms, and particular preference is given to fungi, such as,for example, Saccharomyces cerevisiae or Pichia pastoris.

[0033] For example, the nucleic acid sequence of the DNA fragment codingfor a secretory hybrid protein (PamHNL5xGOX) with HNL activity, whichnucleic acid sequence comprises sequences of the P. amygdalus HNL5 geneand the Aspergillus niger glucose oxidase gene, is depicted in FIG. 4.The amino acid sequence of the PamHNL5xGOX hybrid protein, derived fromthe nucleotide sequence (FIG. 4), is depicted in FIG. 5.

[0034]FIG. 6 shows the comparison of the amino acid sequences of thePrunus amygdalus HNL5 protein and the PamHNL5xGOX hybrid protein.

[0035] In order to obtain the recombinant proteins of the invention, theclones, for example, which show homologies with the known genes of P.amygdalus MDL1 and/or with P. serotina mdl1, mdl2, mdl3, mdl4 and mdl5are treated further.

[0036] In order to obtain recombinant proteins with hydroxynitrile lyaseactivity, the appropriate genes, for example the HNL5 gene, areincorporated, for example, into an expression vector for Pichia pastorisor for Saccharomyces cerevisiae.

[0037] The genomic DNA may be spliced beforehand by means of PCR.Preference is given to preparing a base fragment for constructingexpression plasmids for heterologous expression of the appropriate genein bacteria and eukaryotes. For this purpose, a plasmid is constructedfrom which it is possible to obtain a DNA fragment coding for a gene ofthe invention for incorporation into various expression vectors bycutting with restriction endonucleases.

[0038] Using the genes of the invention, it is thus possible to producea functional HNL by expression in a heterologous host, using, forexample, an inducible promoter system or a constitutively expressedpromoter system.

[0039] It is thus possible to find, out of a large number oftransformants, recombinant strains of, for example, Pichia pastoris,which overexpress recombinant protein with R-HNL activity. Afterinducing expression, the majority of the protein with R-HNL activity canbe found in said strains in the culture supernatant.

[0040] Thus it was possible for the first time to prepare a recombinantprotein which has an R-HNL activity comparable to that detectable inalmonds (Prunus amygdalus kernels) in amounts usable for industrialapplications.

[0041] Unexpectedly it was found that the recombinant proteins withR-HNL activity, which are derived, for example, from the HNL1-5 genes ofthe invention and are denoted Pam-HNL1-5, have, compared with the enzymepreparation isolated from almonds, substantially better properties asbiocatalysts in reaction mixtures for the preparation of cyanohydrinsand can thus be used particularly advantageously for biocatalyticsynthesis of cyanohydrins.

[0042] Furthermore, it is also possible to truncate the sequences of therecombinant proteins of the invention at the C-terminal end or toreplace the sequences in the N- and C-terminal region by those of arelated protein with different functions. Accordingly, the inventionalso relates to proteins altered in this way.

[0043] The recombinant proteins are distinguished in particular also byhaving a host-specific glycosylation, as a result of which therecombinant proteins are substantially more stable than the nativeproteins.

[0044] A particular advantage of the recombinant proteins of theinvention results from the substantially higher stability which causes asubstantially lower amount of enzyme compared with the native enzyme tobe required, in order to achieve high enantiomeric purity. Thus,comparative experiments show that, when using the recombinant proteinsof the invention, merely a tenth of the required amount of nativeprotein is required in order to achieve comparable enantiomeric purities(ee values).

[0045] Accordingly, the invention further relates to the use of therecombinant proteins of the invention (HNLs) for preparing (R)- or(S)-cyanohydrins.

[0046] The starting materials used for preparing the (R)- or(S)-cyanohydrins are an aldehyde or a ketone as substrate, a cyanidegroup donor and a recombinant protein of the invention.

[0047] Aldehydes mean in this connection aliphatic, aromatic orheteroaromatic aldehydes. Aliphatic aldehydes mean in this connectionsaturated or unsaturated, aliphatic, straight-chain, branched or cyclicaldehydes. Preferred aliphatic aldehydes are straight-chain aldehydeswith in particular 2 to 30 carbon atoms, preferably from 2 to 18 carbonatoms, which are saturated or mono- or polyunsaturated. In thisconnection, the aldehyde may have both C-C double bonds and C-C triplebonds. The aliphatic, aromatic or heteroaromatic aldehydes mayfurthermore be unsubstituted or substituted with groups inert under thereaction conditions, for example with unsubstituted or substituted arylor heteroaryl groups, such as phenyl, phenoxy or indolyl groups, withhalogen, hydroxy, hydroxy-C₁-C₅-alkyl, C₁-C₅-alkoxy, C₁-C₅-alkylthio,ether, alcohol, carboxylic ester, nitro or azido groups.

[0048] Examples of aromatic or heteroaromatic aldehydes are benzaldehydeor differently substituted benzaldehydes such as, for example,3,4-difluorobenzaldehyde, 3-phenoxybenzaldehyde,4-fluoro-3-phenoxybenzaldehyde, hydroxybenzaldehyde,methoxybenzaldehyde, furthermore furfural, methylfurfural,anthracene-9-carbaldehyde, furan-3-carbaldehyde, indole-3-carbaldehyde,naphthalene-1-carbaldehyde, phthaldialdehyde, pyrazole-3-carbaldehyde,pyrrole-2-carbaldehyde, thiophene-2-carbaldehyde, isophthalaldehyde orpyridinaldehydes, thienylaldehydes etc.

[0049] Ketones are aliphatic, aromatic or heteroaromatic ketones inwhich the carbonyl carbon atom is substituted unequally. Aliphaticketones mean saturated or unsaturated, straight-chain, branched orcyclic ketones. The ketones may be saturated or mono- orpolyunsaturated. They may be unsubstituted or substituted with groupsinert under reaction conditions, for example with unsubstituted orsubstituted aryl or heteroaryl groups such as phenyl or indolyl groups,with halogen, ether, alcohol, carboxylic ester, nitro or azido groups.

[0050] Examples of aromatic or heteroaromatic ketones are acetophenone,indolylacetone, etc.

[0051] Aldehydes and ketones which are suitable according to theinvention are known or can be prepared as usual.

[0052] The substrates are converted in the presence of the HNLs of theinvention using a cyanide group donor.

[0053] Suitable cyanide group donors are hydrocyanic acid, alkali metalcyanides or a cyanohydrin of the general formula I

R₁R₂C(OH) (CN).

[0054] In the formula I, R₁ and R₂ are independently of one anotherhydrogen or an unsubstituted hydrocarbon group, or R₁ and R₂ aretogether an alkylene group having 4 or 5 carbon atoms, where R₁ and R₂are not simultaneously hydrogen. The hydrocarbon groups are aliphatic oraromatic, preferably aliphatic groups. R₁ and R₂ are preferably alkylgroups having 1-6 carbon atoms, and very preferably the cyanide groupdonor is acetonecyanohydrin.

[0055] The cyanide group donor may be prepared according to knownmethods. Cyanohydrins, in particular acetonecyanohydrin, may also beobtained commercially. Preference is given to using hydrocyanic acid(HCN), KCN, NaCN or acetonecyanohydrin as cyanide group donor, andparticular preference is given to hydrocyanic acid.

[0056] In this connection, it is also possible to liberate hydrocyanicacid from one of its salts such as, for example, NaCN or KCN just priorto the reaction and to add it to the reaction mixture undissolved or insoluble form.

[0057] The conversion may be carried out in an organic, aqueous or2-phase system or in emulsion. The aqueous system used is an aqueoussolution containing the inventive HNL or a buffer solution. The examplesthereof are Na citrate buffer, phosphate buffer, etc.

[0058] Organic diluents which may be used are aliphatic or aromatichydrocarbons which are not or negligibly water-miscible and which areunhalogenated or halogenated, alcohols, ethers or esters or mixturesthereof. Preference is given to using methyl tert-butyl ether (MTBE),diisopropyl ether, dibutyl ether and ethyl acetate or a mixture thereof.

[0059] In this connection, the HNLs of the invention may be presenteither as such or immobilized in the organic diluent, but the conversionmay also be carried out in a two-phase system or in emulsion usingnonimmobilized HNL.

EXAMPLE 1 Isolation of Genomic DNA from Almonds (Prunus amygdalusKernels)

[0060] Dried almonds (Farmgold, batch number L4532, 1999 harvest) werefinely chopped using a knife and frozen in a mortar with liquid nitrogenand ground using a pestle under liquid nitrogen to give a fine powder.0.1 gram of frozen almond powder was directly admixed with 65° C. warm“breaking puffer” (100 mM NaAc; 50 mM EDTA; 500 mM NaCl, adjusted to pH5.5; 1.4% SDS and 20 μg/ml RNAse A). After stirring for 15 minutes, theinsoluble cellular residues were removed by centrifugation (10 min at7000 g) and the supernatant was admixed with the same volume of 10 Mammonium acetate and then incubated on ice for 10 min. Aftercentrifugation at 10,000 g for 15 minutes, the supernatant was extracted2× with phenol/chloroform (1/1, phenol equilibriated with 50 mM Tris, pH8.0). After another extraction with twice the volume ofchloroform/isoamyl alcohol (24/1), the DNA was precipitated from thesupernatant with the same volume of isopropanol, removed bycentrifugation, and the DNA pellet was washed with 70% ethanol and driedin air. The DNA was then dissolved in 200 μl of water at 68° C. for 20min and purified by ethanol precipitation (Ausubel et al., 1999). Afterthe centrifugation, the DNA pellet was dried in air and dissolved in 50μl of water.

EXAMPLE 2 Amplification and Cloning of a Genomic DNA Section of Almond(Prunus amygdalus) DNA Homologous to known Rosaceae mdl Genes

[0061] Since it was known that a plurality of hydroxynitrile lyaseisoenzymes whose sequences are highly homologous to one another canappear in Prunus species (Hu and Poulton, 1999), gene-specific PCRprimers based on sequence homology of the Prunus serotina mdl5 gene andthe Prunus amygdalus MDL1 gene (Suelves et al., 1998) were prepared:

[0062] Primer 1: 5′-CGGAATTCACAATATGGAGAAATCAACAATGTCAG-3′

[0063] Primer 2: 5′-CGGAATTCTTCACATGGACTCTTGAATATTATG-3′

[0064] The amplification was carried out in a 50 μl mixture with 1.2 Uof “Hotstar” Taq DNA polymerase (Qiagen, Hilden, Germany), with 50 ng ofgenomic almond DNA as template, in each case 200 ng of primers 1 and 2,5 μl of a dNTP (2 mM each) mix, all of these in 1× PCR buffer accordingto the “Hotstar Kit” manual (Qiagen, Hilden, Germany), starting with adenaturation step of 15 minutes at 95° C., followed by 30 cycles (1 min95° C., 30 sec 64° C., 1 min 72° C.) for amplification and a finalincubation at 72° C. for 5 min for preparation of complete products.

[0065] Said PCR produced a DNA fragment of approx. 2.16 kb in size(determined by analysis by means of agarose gel electrophoresis). ThisPCR product was purified by means of the “Qiaquick Kit” (Qiagen, Hilden,Germany) according to the enclosed manual and sequenced using the “DyeDeoxy Terminator Cycle Sequencing” kit (Applied Biosystems Inc., ForsterCity, Calif., USA) according to the primer walking strategy startingfrom the two primers used for the PCR. The obtained DNA sequence of thePCR fragment of 2162 base pairs total length is depicted in FIG. 1.

[0066] Approx. 0.5 μg of the purified PCR product was cut withrestriction endonuclease EcoRI and cloned into plasmid vector pBSSK(−)(Stratagene Cloning Systems, La Jolla, Calif., USA) via the EcoRIcleavage site. The insert of a resultant recombinant molecule (thecorresponding plasmid was denoted pBSPamHNL5g) was sequenced accordingto the method described above, and the sequence of the cloned fragmentwas 100% identical to the sequence of the above-described PCR productobtained with the two primers (1 & 2).

EXAMPLE 3 Sequence Analysis of the Genomic Prunus amygdalus DNA Fragmentobtained by PCR Amplification with primers 1 and 2

[0067] In the region of the PCR-amplified and sequenced DNA section, anopen reading frame which is interrupted by 3 introns was found. Saidthree introns were identified with the aid of the “GT . . . AG” intronconsensus sequence. The reading frame starts with an ATG codon atposition +13 and ends with a stop codon at position +2151.

[0068] For the coding region, the fragments of positions 13 to 115 (exonI), positions 258 to 917 (exon II), 1121 to 1961 (exon III) and 2078 to2150 (exon IV) were joined together. The assembled DNA sequence codesfor a protein with 559 amino acids and a calculated molecular weight of61 kDa or 57.9 kDa for an N-terminally processed form. The peptidemasses were calculated with the aid of the GCG program package (GeneticsComputer Group, Wisconsin, USA). Said protein was denoted PamHnI5.

[0069] The protein sequence derived for the open reading frame (withoutintrons) is shown in FIG. 3. It was possible to determine distinctivehomologies to known Rosaceae hydroxynitrile lyases (Blast program, GCGpackage, version 10.1, Genetics Computer Group, Wisconsin, USA), thehighest homologies being to the published sequences of Prunus amygdalusMdl1 (99 percent identical, Suelves et al. 1998) and of Prunus serotinaMdl5 (94 percent identical, Hu and Poulton, 1999). With the aid of thishomology, a cleavable signal sequence with cleavage between S27 and L28was identified. It was possible to detect a cleavage site of this typein the two Prunus serotina Mdl1 and Mdl4 isoenzymes by N-terminalsequencing of the native proteins purified from plant material (Zihua Huand Jonathan E. Poulton, Plant Physiology, 119, 1535-1546, 1999). Thesequences of various HNL isoenzymes present in Rosacea species are knownonly for Prunus serotina. Due to the highest homologies to the Prunusserotina Mdl5 sequence, the new HNL gene from Prunus amygdalus wasestablished as HNL5. Searching for sequence motifs in the PROSITEsequence motif database (GCG package, version 10.1, Genetics ComputerGroup, Wisconsin, USA) revealed the presence of 13 potentialN-glycosylation sites (drawn into FIG. 3).

EXAMPLE 4

[0070] Obtaining an intron-free Prunus amygdalus HNL5 gene by PCRsplicing. By means of a specific PCR strategy using overlapping primers,the coding regions were linked to one another (according to FIG. 2) by 4successive PCR reactions.

[0071] In the first round of PCR (PCR1-1 and PCR1-2) exons II and IIIwere amplified using the primer pairs PamHNL5b/PamHNL5c (PCR1-1) andPamHNL5d/PamHNL5e (PCR1-2), respectively. The 50 μl PCR mixtures in 1×PCR buffer (Qiagen) contained: in each case 100 pmol of the appropriateprimers, 2.5 U of “Hotstar” Taq DNA polymerase (Qiagen), 5 μl of a dNTP(2 mM each) mix, 10 ng of plasmid pBSPamHNL5g as template. The followingprogram was run: 15 min 95° C., 30 cycles 1 min 95° C., 30 sec 68° C., 1min 72° C., then finally 5 min 7° C. for preparation of completeproducts). After electrophoretic separation in an agarose gel, theproducts from PCR1-1 and PCR1-2 were eluted from the gel by means of theQiaexll kit.

[0072] Amplification of approx. 50 ng of the product from PCR1-1 with ineach case 100 pmol of primers PamHNL5a2 and PamHNL5c led to an extensionof said first PCR product in the second round of PCR (PCR2). Thefollowing program was run: 15 min 95° C., 30 cycles 1 min 95° C., 30 sec68° C., 1 min 72° C., then finally 5 min 72° C. for preparation ofcomplete products). The other conditions were the same as for PCR1.After electrophoretic separation, this PCR product was likewise purifiedvia an agarose gel and eluted.

[0073] In the third round of PCR (PCR3), the products from PCR1-2 andPCR2 were linked to one another by primer-less PCR with the aid of theoverlapping ends (5 cycles of 1 min at 94° C., 30 sec at 68° C. and 1.5min at 72° C., in each case approx. 100 ng of the two products fromPCR1-1 and PCR2 in 50 μl mixtures in 1× PCR buffer (Qiagen), 5 μl of thedNTP (2 mM each) mix and 2.5 U of “Hotstar” Taq DNA polymerase (Qiagen).

[0074] The full length of the coding Prunus amygdalus hn15 gene wascompleted and the complete product amplified in a fourth round of PCR(PCR4) using in each case 100 pmol of primers PamHNL5al and PamHNL5f.Said primers were directly added to the PCR 3 reaction mixture. Thefollowing program was run: 20 cycles of 1 min 95° C., 30 sec 63° C., 1.5min 72° C. and, finally, 5 min 72° C.). The other conditions were thesame as for PCR1.

[0075] The product obtained in the final round of PCR was fractionatedvia a preparative agarose gel and DNA of 1.6-1.8 kb in size was elutedfrom the gel by means of the “Quiaexll” kit (Qiagen) and cloned intoplasmid pBSSK(−) via the EcoRI cleavage site. A clone having the correctrestriction pattern was selected and sequenced.

[0076] The sequence in the coding region was 100% identical to the exonsof the genomic DNA sequence. This clone was denoted pBSPamHNL5orf.

[0077] Oligonucleotide Primers PamHnl5a15′-GAAGATCTGAATTCCATGGAGAAATCAACAATGTCAGTTATACTATTTGTGTTGCATCTTCTTG-3′PamHnl5a25′-CTATTTGTGTTGCATCTTCTTGTTCTTCATCTTCAGTATTCAGAGGTTCACTCGCTTGCCAATACTTC-3′PamHnl5b5′-GTTCACTCGCTTGCCAATACTTCTGCTCATGATTTTAGCTACTTGAAGTTTGTGTACAACGCCACTG-3′PamHnl5c 5′-GATGTATTGGAAGAGAAGAGGATCTTCTCTACT-3′ PamHnl5d5′-GATCCTCTTCTCTTCCAATACATCAAATTTGTCAGCTATTGGAGTCATATATACGG 3′ PamHnl5e5′-CAACCGGATTGACCTTTCTTGCAGGATTTGAAGGCCCACATACCTTCCTAACATCAGATAGAAGCC-3′PamHnl5f5′-GAAGATCTGGAATTCTTCACATGGACTCTTGAATATTATGAATAGCCTCCAACCGGATTGACCTTTCTTGCAG-3′

EXAMPLE 5

[0078] The preparation of a base fragment for constructing expressionplasmids for heterologous expression of the Prunus amygdalus HNL5 genein bacteria and eukaryotes.

[0079] The aim of this experiment was to construct a plasmid from whicha DNA fragment coding for Prunus amygdalus HNL5 for incorporation intovarious expression vectors can be obtained by restriction endonucleasecleavage. In this connection, PCR amplification added suitable sequencesto the ends of the Prunus amygdalus HNL5 gene contained in pBSPamHNL5orfvia appropriate primers.

[0080] The insert of plasmid pBSPamHNL5orf was amplified by means of PCRusing the primers PCRHNL5-a and PCRHNL5-e (10 ng of DNA of plasmidpBSPamHNL5orf as template, 400 ng of primer PCRHNL5-a, 200 ng of primerPCRHNL5-e). The PCR reaction was carried out in 50 μl mixtures in 1× PCRbuffer (Qiagen), containing 5 μl of the dNTP (2 mM each) mix and 1.2 Uof “Hotstar” Taq DNA polymerase (Qiagen). The following program was run:15 min 95° C., 30 cycles 1 min 95° C., 30 sec 68° C., 1.5 min 72° C.,then finally 5 min 72° C. for preparation of complete products).

[0081] After cutting with restriction endonuclease EcoRI, the DNAfragment obtained was cloned into vector PBSSK(−) (Stratagene, USA) andverified by sequencing. The resultant plasmid was called pBSPamHNL5ex.

[0082] Oligonucleotide Primers: PCRHNL5-a5′-TCGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGAGAAATCAACAATGTCAGTTATACTATTTGTGTTGCATC-3′ PCRHNL5-e5′-CGAATTCGCCCTTTCGCATGCTCACATGGACTCTTGAATATTATGAATAGCCTC-3′

EXAMPLE 6 Construction of Expression Constructs for HeterologousExpression of the HNL5 Gene in Pichia pastoris

[0083] DNA of plasmid pBSPamHNL5ex was cut with EcoRI, and the HNLfragment was separated from the vector part by means of preparative gelelectrophoresis. After eluting the HNL fragment DNA by means of Qiaex IIkit (Qiagen), said fragment was cloned into plasmids pHILD2 and pGAPZ(Invitrogen, San Diego, Calif., USA) via the EcoRI cleavage sites. Thecorrect orientation of the insert toward the promoters was checked withthe aid of control cuts. In each case, one clone having a correctlyorientated insert was selected and preserved. The correct transitionsfrom vector part to incorporated HNL fragment were verified bysequencing. Said two plasmids for expression in Pichia pastoris formedwere denoted pHILDPamHNL5a (for inducible expression) and pGAPPamHNL5a(for constitutive expression).

EXAMPLE 7 Inducible Expression of the Prunus amygdalus HNL5 Gene inPichia pastoris

[0084] DNA of plasmid pHILDPamHNL5a was cut with restrictionendonuclease Not1 and transformed into Pichia pastoris GS115(Invitrogen, San Diego, Calif., USA). Transformation was carried outaccording to the protocols of the Pichia expression kit (Invitrogen, SanDiego, Calif., USA). 100 histidine-prototrophic clones were cultivatedin liquid medium (500 ml shaker cultures) according to the protocols ofthe Pichia expression kit and induced with methanol for 48 hours. Thecells were removed by centrifugation and suspended with disruptionbuffer (50 mM Tris-HCl pH 7.6) to an optical density (OD₆₀₀) of 50.0 anddisrupted in a “Merckenschlager” homogenizer (Braun, Melsungen, FRG)according to the “glass bead method” (Ausubel et al., Current Protocolsin Molecular Biology, Green Publishing Associates andWiley-Interscience, New York, 1999). Both the culture supernatants andcell lysates were assayed for HNL enzyme activity using a standardactivity assay (analogous to WO 97/03204) and using racemicmandelonitrile as substrate. Two clones which showed, in these shakerflask experiments, the best activities in the culture supernatant(Pichia pastoris PamHNL5-a37 and Pichia pastoris PamHNL5-a4) wereselected and preserved. Analysis of the methanol utilization typeresulted in the phenotype Mut

(good methanol utilization) for Pichia pastoris PamHNL5-a37 and thephenotype Mut^(s) (slow methanol utilization) for Pichia pastorisPamHNL5-a4.

EXAMPLE 8 Constitutive Expression of the Prunus amygdalus HNL5 Gene inPichia pastoris

[0085] Plasmid pGAPPamHNL5a was transformed into P. pastoris GS115.Transformation was carried out according to the protocols of the Pichiaexpression kit from Invitrogen Corp (San Diego, Calif., USA).Transformants were selected from YPD complete medium plates with 100mg/l Zeocin. 100 Zeocin-resistant clones were cultivated in in each case500 ml of YPD complete medium and incubated with shaking at 30° C. for96 hours. The cells were removed by centrifugation and HNL activity wasdetermined in the culture supernatant. A clone which showed the bestactivity in the culture supernatant was preserved and denoted Pichiapastoris PamHNL5-a2.

EXAMPLE 9 HNL Production in a Laboratory Bioreactor using a Pichiapastoris Strain Transformed with the Prunus amygdalus HNL5 Gene(Methanol-Inducible Expression System)

[0086]Pichia pastoris PamHNL5-a37 was grown in a standard laboratorybioreactor (total volume 42 1) in a three-phase process. Said processconsisted of a first exponential and a second linear growth phase forbiomass formation and a subsequent expression phase for formation of therecombinant Prunus amygdalus HNL enzyme, in principle following themethod described for the production of recombinant Hevea brasiliensisHNL (Hasslacher, M., Schall, M., Hayn, M., Bona, R., Rumbold, K., Lückl,J., Griengl, H., Kohlwein, S. D., Schwab, H.: High level intracellularexpression of hydroxynitrile lyase from the tropical rubber tree Heveabrasiliensis in microbial hosts. Protein Expression and Purification 11,61-71, 1997).

[0087] In detail, the following conditions were kept to: Chemicals1.-8., measured for 20 liters and dissolved in 15 liters of water, wereinitially introduced into the bioreactor and sterilized together withthe reactor at 121° C. for 1 hour. After cooling to 29° C., the pH ofthe medium was adjusted to pH 5.0 with ammonia (chemical 9, initiallyintroduced in a sterile feed bottle). Subsequently, approx. 200 ml of asterile-filtered trace element solution (chemicals 10-18, appropriateamounts for 20 l) were introduced into the bioreactor via a feed bottle.

[0088] The bioreactor prepared in this way was inoculated with 2 litersof preculture which had been cultivated in shaker flasks at 30° C.according to the conditions stated in the manual of the Pichiaexpression kit (Invitrogen Corp., San Diego, Calif., USA). Culturing wascarried out at a constant temperature of 29° C. Controlling aeration(between 15 and max. 40 liters of air/min) and stirrer revolutions(between 250 to 500 rpm) maintained the O₂ partial pressure at a valueabove 30% of the saturation concentration. After 21 hours, the biomasshad grown to a value in the region of 22 g/l of dry cell mass (DCM).From this time onward, sterile glycerol was metered in in constant smallportions at 15 min intervals, and 130 g of glycerol were added per hour.In this, second linear growth phase, it was possible to reach a biomassconcentration in the range of 70 g/l DCM over a period of 42 hours.

[0089] Subsequently, the third phase was initiated by inducingexpression via metering in methanol. In this connection, the methanolcontent of the culture broth was adjusted to a value of 0.8-1% byweight. At the start of and after two days of induction, in each caseanother portion of sterile trace element solution (chemicals 10-18,appropriate amounts for 20 l, dissolved in approx. 200 ml of water) wasadded. After an induction phase of 110 hours, it was possible to obtainan amount of enzyme of 110 U/ml of culture broth. After removing thecells, for example by centrifugation, it is possible to obtain a crudeenzyme preparation which can be used directly for biocatalyticconversions.

[0090] The following chemicals were used for preparing the culturemedium (amount per liter): 1. 85% ortho-phosphoric acid 21 ml 2. CaSO₄0.9 g 3. K₂SO₄ 14.3 g 4. MgSO₄.7H₂O 12.2 g 5. KOH (chemicals 1 to 5 inanalytical grade) 6. Glycerol, technical grade 50 ml 7. Deionized water,home grade, 5.5-9.1 μS/cm conductivity 8. Anti-foaming agent 10% Acepol83E 1 ml (Carl Becker Chemie GmbH, Hamburg, Germany) 9. 25% ammonia,technical grade

[0091] Trace elements and vitamin H (all chemicals in analytical grade):10. Biotin 0.8 mg 11. CuSO₄.5H₂O. 24.0 mg 12. Kl 0.32 mg 13. MnSO₄.H₂O12.0 mg 14. Na₂MoO₄.2H₂O 0.2 mg 15. H₃BO₃ 0.08 mg 16. CoCl₂ 2.0 mg 17.ZnSO₄.7H₂O 80 mg 18. Fe(II)SO_(4.)7H₂O 260 mg

EXAMPLE 10

[0092] Construction of a clone for expressing the Prunus amygdalus HNL5gene as fusion protein with N-terminal and C-terminal parts ofAspergillus niger glucose oxidase.

[0093] This construct was designed such that the fusion protein formedis directed into the secretory pathway via the heterologous signalsequence.

[0094] In a PCR in a 50 μl mixture in 1× PCR buffer (Qiagen), with ineach case 100 pmol of primers Glucox2 and Glucoxct, 10 ng of plasmidpPamHNL5orf as template, 5 μl of dNTP (2 mM each) mix, and 1.2 U of“Hotstar” Taq polymerase (Qiagen), the C-terminal and N-terminal ends ofthe HNL5 gene were replaced by a sequence derived from glucose oxidaseand truncated, respectively (program: 15 min 95° C., 30×: 1 min 95° C.,1 min 68° C., 2 min 72° C., and finally 10 min 72° C). Finally, in a2^(nd) PCR in a 50 μl mixture in 1× PCR buffer (Qiagen) with 0.1 μl ofproduct from the first PCR as template, in each case 100 pmol of primersGlucoxl and Glucoxct, 2 μl of dNTP (2 mM each) mix, and 2.5 U of Pwopolymerase (Roche Diagnostics, Mannheim, Germany), (program: 5 min 95°C., 30 times: 1 min 95° C., 0.5 min 68° C., 3 min 72° C. and finally 3min 72° C) the 5′ region of the gene was completed.

[0095] The PCR product was incorporated into plasmid pHILD2 (Invitrogen,San Diego, Calif., USA) after cutting with EcoRI, via EcoRI cleavagesites simultaneously introduced at the ends of the DNA fragment. A clonehaving the correct orientation of the insert toward the aox promoter ofplasmid pHILD2 was verified by sequencing the transition regions fromvector to insert and preserved. The plasmid constructed in this way wasdenoted pHILDPamHNL5gox.

[0096] Not1-linearized DNA of plasmid pHILDPamHNL5gox was transformedinto the strain Pichia pastoris GS115 and also into theprotease-deficient strain Pichia pastoris SMD1168. From each mixture,several histidine-prototrophic clones were cultured in shaker flasks andHNL activity was determined in the cultured supernatant (standardassay). These experiments were carried out analogously, as described inexample 7. It was possible to find in the culture supernatant of someclones HNL activity and thus to state that the signal sequence of theAspergillus niger gox gene is capable of directing the heterologous HNL5protein, when expressed in Pichia pastoris, into the secretory pathway.

[0097] Oligonucleotide Primers Used GLUCOX15′-CACGAATTCATCATGCAGACTCTCCTTGTGAGCTCGCTTGTGGTCTCCCTCGCTGCGGCCCTGCCACACTAC-3′GLUCOX25′-TGCGGCCCTGCCACACTACATCAGGAGCAATGGCATTGAAGCCTACAACGCCACTGATACAAGCTCGGAAGGATC-3′GLUCOXCT 5′-GAATTCGCATGCGGCCGCTCACTGCATTGACCTTTCTTGCAGGATTTGAAG-3′

[0098] The nucleic acid sequence of the DNA fragment for a secretoryhybrid protein (PamHNL5xGOX) with HNL activity is depicted in FIG. 4,and the amino acid sequence derived therefrom is represented in FIG. 5.A comparison of the amino acid sequences of Prunus amygdalus HNL5 andthe hybrid protein PamHNL5xGOX can be found in FIG. 6.

EXAMPLE 11

[0099] Recombinant protein with HNL activity, which had been producedusing the recombinant strain Pichia pastoris PamHNL5-a37, as describedin example 9, was subjected to a glycosylation analysis byendoglycosidase digest. For this purpose, 100 ml of the culturesupernatant were 10 times concentrated by ultrafiltration (Biomax 30,000NMWL, Millipore, Bedford, Mass., USA). Samples 1-2 were treated withN-glycosidase F (N-glycosidase F kit, Roche Diagnostics, Mannheim, D).

[0100] For samples 4-5, an HNL preparation from almonds from Roche wasused, and for samples 6 and 7 the concentrated culture supernatant wastreated with endoglycosidase H (Roche Diagnostics, Mannheim, D). Allmixtures were carried out in a total volume of 10 μl.

[0101] Std.

[0102] Molecular weight standard from N-glycosidase F kit (5 μl=5 μg)

[0103] Sample 1:

[0104] 2 U of PamHNL5 treated with 2.4 U of enzyme according to theprotocol of the kit.

[0105] Sample 2:

[0106] 2 U of PamHNL5 treated with 2.4 U of enzyme according to theprotocol of the kit, but without denaturation buffer and without heatdenaturation

[0107] Sample 3:

[0108] 2 PamHNL5 without treatment

[0109] Sample 4:

[0110] 0.25 U of Roche R-HNL preparation grade III from almonds (10.3U/mg), treated with 2.5 U of N-glycosidase F according to the protocolof the kit

[0111] Sample 5:

[0112] 0.25 U of the Roche preparation grade III (10.3 U/mg), untreated

[0113] Sample 6:

[0114] 2.4 U of PamHNL5 were incubated with 50 mU of endoglycosidase Hin 20 mM phosphate buffer without denaturation at 37° C. for 12 hours.

[0115] Sample 7:

[0116] 2.4 U of PamHNL5 were incubated with 50 mU of endoglycosidase Hin 20 mM phosphate buffer, 0.2% SDS, 0.4% mercaptoethanol at 37° C. for12 hours.

[0117] After treatment with the glycosidases, the samples were separatedon a 12 percent strength SDS polyacrylamide gel and stained withCoomassie Blue.

[0118] These results (see FIG. 7) show that a large part of theoligosaccharides bound to PamHNL5 can be removed by endoglycosidase Heven without denaturation of the PamHNL5 protein.

[0119] Cleaving off the oligosaccharides leads from a protein smearvisible around sizes of from 70 to over 100 kDa to a sharp band at about60 kDa, corresponding to the calculated molecular weight of anonglycosylated PamHNL5 protein.

[0120] A comparable protein band is not present in the Roche preparationor present only to a negligible extent. In addition, it is impossible tosee a significant difference between an untreated protein preparationand a preparation treated with endoglycosidase F. From this finding, itcan definitely be stated that the recombinant PamHNL5 enzyme iscompletely different from the enzyme material obtained from almonds.

EXAMPLE 12 Cloning of a genomic DNA fragment having the Coding Region ofthe Prunus amygdalus HNL1 Gene

[0121] A genomic DNA fragment having the coding region of the Prunusamygdalus HNL1 gene was amplified from genomic almond DNA (preparation,see example 1) with the aid of a PCR using primers mandlp2f(5′-ACTACGAATTCGACCATGGAGAAATCAAC-3′) and ecpamHNL1e(5′-CAGAATTCGCCCTTGTGCATGCATCGATTAAAGAACCAAGGATGCTGCTGA C-3′)

[0122] The amplification was carried out in 50 μl reactions with 1.2units of “Hotstar” DNA polymerase (Qiagen GmbH, Hilden, Germany), ineach case 10 pmol of the two primers, 2 μl of a dNTP mix (5 mM each) and100 ng of genomic almond DNA in standard PCR buffer (Qiagen GmbH,Hilden, Germany). The following PCR program was used: 15 min 95° C.,then 10 cycles of 1 min at 94° C., 1 min 45° C. and 1 min 20 sec at 72°C., followed by 30 cycles of 1 min at 94° C., 1 min at 64° C. and 1 min20 sec at 72° C. and a final extension step at 72° C. for 5 min.

[0123] Analysis of the DNA obtained showed that this PCR produced aplurality of DNA fragments of different sizes. Amplified DNA wasseparated in a preparative agarose gel. DNA from a band of the size tobe expected for the HNL1 gene of approx. 2.1 kb was isolated from saidagarose gel (Qiaquick Gel Extraction Kit, Qiagen GmbH, Hilden, Germany).The DNA obtained, after digest with restriction endonuclease EcoRI, wascloned into cloning vector pBSSK(−) (Stratagene Cloning Systems, LaJolla, Calif., USA) via the EcoRI cleavage site. 5 clones withappropriate inserts were isolated and the inserts were sequenced bymeans of the primer walking strategy. A clone corresponding to theconsensus sequence obtained in this way was selected and the containedplasmid was denoted pSKpamHNL1_(—)5_(—)3.

[0124] The DNA sequence of the Prunus amgdalus HNL1 gene was verifiedand finally determined by amplifying another genomic DNA fragment usingprimers mandlp3f (5′ACTACGAATTCGACCATGGAGAAATCAACAATG-3′) and pamHNL1end(5′-ATGCTGCTGACTTGAGGGAATC-3′). The amplification was carried out in 50μl reactions with 2.5 units of “Hotstar” DNA polymerase (Qiagen GmbH,Hilden, Germany), in each case 10 pmol of the two primers, 2 μl of adNTP mix (5 mM each) and 50 ng of genomic almond DNA in standard PCRbuffer (Qiagen GmbH, Hilden, Germany). The following PCR program wasused:

[0125] 15 min 95° C., then 5 cycles of 1 min at 94° C., 30 sec at 55° C.and 2 min at 72° C., then 30 cycles with 1 min at 94° C., 30 sec at 68°C. and 2 min at 72° C. and a final extension step at 72° C. for 7 min.

[0126] After fractionation of the PCR product in an agarose gel, asingle DNA band was detected.

[0127] The PCR product was purified by means of the Qiaquick GelExtraction Kit (Qiagen GmbH, Hilden, Germany) and directly sequenced bymeans of the primer walking strategy. The DNA sequence is depicted inFIG. 8. Possible introns were identified by their general 5′ and 3′splice sites and their homology to the Prunus serotina HNL1 gene. Thenucleotides in the regions of the three introns are shown in lower caseletters in order to recognize the intron regions. The cloned insert inplasmid pSKpamHNL1_(—)5_(—)3 has the same sequence. The protein sequenceof the Prunus amygdalus HNL1 protein was derived from the coding regionof the DNA sequence and is depicted in FIG. 9.

EXAMPLE 13

[0128] 0.5 g (3.9 mmol) of octanal was dissolved in 6 ml of tert-butylmethyl ether and 7.5 ml of an aqueous enzyme solution with recombinantR-HNL from example 9 (pH 3.8) were added. After addition of 0.33 ml (8.4mmol) of hydrocyanic acid, the mixture was vigorously stirred on amagnetic stirrer at room temperature in order to form an emulsion, andthe reaction was followed by means of GC on a cyclodextrin column. Aftera reaction time of 3 hours, cyanohydrin was formed with 81.1%ee and 48%conversion.

EXAMPLE 14

[0129] 80 ml (34 units/mmol aldehyde) of an aqueous enzyme solution withrecombinant R-HNL (34 units/mmol aldehyde) were diluted with 10 ml of200 mM potassium phosphate/sodium citrate buffer pH 3.8 and added to asolution of 42.2 g (300 mmol) of 2-chlorobenzaldehyde and 42 ml oftert-butyl methyl ether, precooled to 1° C. Subsequently, 19.6 ml (501mmol) of hydrocyanic acid were metered into the reaction mixture withstirring at 950 rpm within 40 min. After derivatization of cyanohydrinwith acetyl chloride, the course of the reaction was followed by meansof GC on a cyclodextrin column. Hours % Conversion % ee 3.5 71.5 90.6 2299.7 90

COMPARATIVE EXAMPLE

[0130] 0.25 to 1 ml of R-oxynitrilase solution (E.C.4.1.2.10; 2187units/ml) was diluted to 4 ml with 50 mM citrate/phosphate buffer (pH4.0), and the pH of the enzyme solution was adjusted to pH 4.0, whereappropriate, with a few drops of citric acid solution. A solution of 3ml of tert-butyl methyl ether and 0.8 g (5.69 mmol) of2-chlorobenzaldehyde was added to said solution and, subsequently, 445μl (11.38 mmol) of hydrocyanic acid were added. The reaction mixture wasstirred at 900 rpm by means of a magnetic stirrer at room temperature.

[0131] Conversion and enantiomeric purity of the (R)-cyanohydrin formedwere analyzed by means of GC. For this purpose, a sample of the reactionsolution was centrifuged and 50 μl of the organic phase were dilutedwith dichloromethane. After derivatization with acetyl chloride, a gaschromatographic analysis on a cyclodextrin column was carried out. 0.25ml of enzyme solution 0.5 ml of enzyme solution 1.0 ml of enzymesolution corresponding to 96 corresponding to 192 corresponding to 384units/mmol of aldehyde units/mmol of aldehyde units/mmol of aldehydeTime (h) % Conversion % ee % Conversion % ee % Conversion % ee 1.5 79.177.5 97.6 81.6 98.7 89.4 3 98 77.4 100 81.5 100 89.1

[0132] The comparative experiment showed that when using the recombinantproteins of the invention in analogy to example 13 only a tenth of therequired amount of native protein is required in order to achievecomparable enantiomeric purities (ee values).

1. A new gene containing a DNA sequence coding for hydroxynitrile lyase,which gene can be prepared via a primer combination based on the DNAsequence of the 5′-region of the mdl genes from Prunus serotina and fromPrunus amygdalus and/or a primer 2 based on the 3′-region of the DNAsequences of one of the hydroxynitrile lyase isoenzymes from Prunusserotina or from Prunus amygdalus, subsequent amplification with a DNApolymerase using a DNA from organisms, containing genes coding forhydroxynitrile lyase, as templates and cloning.
 2. The new gene asclaimed in claim 1, which can be prepared from primers based on thesequences of the Prunus amygdalus MDL1 gene and of one of the Prunusserotina mdl genes, and the subsequent amplification and cloning.
 3. Thenew gene as claimed in claim 1, which can be prepared from primers basedon the sequences of the Prunus serotina mdl5 gene and of the Prunusamygdalus MDL1 gene, subsequent amplification and cloning, which genehas the nucleotide sequence depicted in FIG. 1 or is at least 80%identical thereto.
 4. The new gene as claimed in claim 1, which can beprepared from primers based on the sequenc of the Prunus serotina mdl1gene, subsequent amplification and cloning, which has the nucleotidesequence depicted in FIG. 8 or is at least 80% identical thereto.
 5. Thenew gene as claimed in claim 1, which has the nucleotide sequencedepicted in FIG. 1 from nucleotide 13 until nucleotide 2151 continuouslyor without the intron regions from nucleotide 116 until 257, 918 until1120 and 1962 until
 2077. 6. The new gene as claimed in claim 1, whichhas the nucleotide sequence depicted in FIG. 8 from nucleotide 1 untilnucleotide 2083 continuously or without the intron regions fromnucleotide 104 until 249, 907 until 1047 and 1889 until
 1993. 7. Arecombinant protein, which can be prepared in suitable host cells byheterologous expression of the DNA sequence of the Prunus amygdalus HNLgenes as claimed in any of claims 1 to
 6. 8. The recombinant protein asclaimed in claim 7, which comprises host-specific glycosylation.
 9. Therecombinant protein as claimed in claim 7, wherein said protein isprepared by expression in a eukaryotic microorganism.
 10. Therecombinant protein as claimed in claim 7, wherein said protein isprepared by expression in a fungus.
 11. The recombinant protein asclaimed in claim 7, wherein the protein has the amino acid sequencederived from the nucleotide sequence of the gene as claimed in claim 3or
 4. 12. The use of a DNA sequence of genes as claimed in claims 1 to6, which codes for the signal peptide of a hydroxynitrile lyase ofRosacea species for secretory expression of heterologous proteins withhydroxynitrile lyase activity in host cells.
 13. A fusion protein orheterologous protein with hydroxynitrile lyase activity which can beprepared by using a DNA sequence of genes as claimed in claim 1 to 6,which codes for the signal peptide of a hydroxynitrile lyase of Rosaceaspecies, and by secretory expression thereof in host cells.
 14. Thefusion protein as claimed in claim 13, wherein the fusion protein hasthe nucleic acid sequence depicted in FIG. 4, comprising sequences ofthe gene as claimed in claim 3 and the Aspergillus niger glucose oxidasegene, and also the amino acid sequence according to FIG. 5, which isderived from said nucleic acid sequence.
 15. The recombinant protein asclaimed in claim 7, which either has been truncated at the C-terminalend or in which the sequences in the N- and C-terminal region have beenreplaced by those of a related protein with different functions.
 16. Theuse of proteins as claimed in any of claims 7-11 or 13-15 for preparing(R)- or (S)-cyanohydrins.
 17. A process for preparing (R)- or(S)-cyanohydrins, which comprises reacting aliphatic, aromatic orheteroaromatic aldehydes and ketones with proteins as claimed in any ofclaims 7-11 or 13-15 in an organic, aqueous or 2-phase system or inemulsion in the presence of a cyanide group donor.